The network of enzymes involved in the ubiquitination (ligases) and deubiquitination (deubiquitinases or DUbs) of cellular proteins is one of the largest in the human proteome and plays a major role in the regulation of cellular homeostasis. This network interacts with other networks in the cell, especially the kinome, in regulating transcriptional activation and protein degradation among other functions. Finally, dysregulation of this network has been linked to a variety of diseases including neurodegenerative diseases, cancer, and arthritis and inflammation. One of the most powerful tools for determining the specificity of proteases has been the use of peptide libraries to define substrate preferences and determine the influence of amino acids flanking the scissile bond on cleavage efficiency. These studies also provide insights into the nature of side chains interacting with binding subsites within the enzyme's active site. Because deubiquitinases hydrolyze isopeptide bonds between ubiquitin (Ub) and the protein substrate, this type of analysis cannot be performed with such linear peptide libraries which do not mimic the geometry of an isopeptide linkage. In this application, we propose to construct a peptide library linked to Ub through a physiologically relevant isopeptide bond between the carboxyl of the C-terminal Gly of Ub and the 5-amine of a lysine residue in the peptide. A microarray of the library will be constructed on a glass substrate to allow facile analysis of the activity of individual DUbs against each Ub-isopeptide in the library. The data generated in this system will allow us to begin to define the substrate selectivity of different DUbs and the influence of different amino acids flanking the isopeptide lysine on catalytic efficiency of the DUbs. These results will help refine the pattern of interactions in the ubiquitin network and their role in regulating cellular physiology. PUBLIC HEALTH RELEVANCE: LifeSensors proposes to develop an innovative series of new tools that will impact the characterization of the ubiquitination/deubiquitination network. This network is intimately involved - both directly and indirectly through its interactions with other regulatory networks - in the maintenance of cellular homeostasis. Dysregulation of specific members of this network has been implicated in the etiology of a variety of human pathologies including neurodegenerative diseases, cancer, and arthritis and inflammation. The tools developed under this grant will provide new methods for understanding the functioning of ubiquitin pathway enzymes and new approaches to the development of therapeutic agents targeting these enzymes.